Alkylating agents, such as drugs derived from nitrogen mustard, that is bis(2-chloroethyl)amine derivatives, are used as chemotherapeutic drugs in the treatment of a wide variety of cancers. Melphalan, or p-bis-(2-chloroethyl)-amino-L-phenylalanine (compound (Id), CAS No. 148-82-3), is an alkylating agent which is a conjugate of nitrogen mustard and the amino acid phenylalanine (U.S. Pat. No. 3,032,584). Melphalan is used clinically in the treatment of metastatic melanomas, but has limited efficacy, dose-limiting toxicities and resistance can develop.

Melphalan flufenamide ethyl ester (L-melphalanyl-L-p-fluorophenylalanine ethyl ester, melflufen, compound (Ib)) is a derivative of melphalan conjugated to the amino acid phenylalanine, creating a dipeptide (WO 01/96367):

The monohydrochloride salt of melflufen (L-melphalanyl-L-p-fluorophenylalanine ethyl ester monohydrochloride; hydrochloride salt of (Ib); CAS No. 380449-54-7) is referred to as melflufen hydrochloride.

When studied in cultures of human tumor cells representing approximately 20 different diagnoses of human cancers, including myeloma, melflufen showed 50- to 100-fold higher potency compared with that of melphalan (http://www.oncopeptides.se/products/melflufen/accessed 26 Mar. 2015). Data disclosed in Arghya, et al, abstract 2086 “A Novel Alkylating Agent Melphalan Flufenamide Ethyl Ester Induces an Irreversible DNA Damage in Multiple Myeloma Cells” (2014) 5th ASH Annual Meeting and Exposition, suggest that melflufen triggers a rapid, robust and irreversible DNA damage, which may account for its ability to overcome melphalan-resistance in multiple myeloma cells. Melflufen is currently undergoing phase I/IIa clinical trials in multiple myeloma.
A process for preparing melflufen in hydrochloride salt form is described in WO 01/96367, and is illustrated in Scheme 1, below. In that process N-tert-butoxycarbonyl-L-melphalan is reacted with p-fluorophenylalanine ethyl ester to give N-tert-butoxycarbonyl-L-melphalanyl-L-p-fluorophenylalanine ethyl ester. After purification by gradient column chromatography the yield of that step is 43%.

As shown in Scheme 1, the known process for preparing melflufen (in hydrochloride salt form) uses the cytotoxic agent melphalan as a starting material, and melflufen is synthesised in a multistep sequence. Melphalan is highly toxic, thus the staring materials and all of the intermediates, and also the waste stream generated, are extremely toxic. That is a major disadvantage in terms of safety, environmental impact and cost when using the process on a large scale. Therefore, an improved and safer method is highly desired, especially for production of melflufen on a large scale. Further, the purity of commercially available melphalan is poor due to its poor stability, the yield in each step of the process is poor, and purity of the final product made by the known process is not high.
A process for preparing melphalan is described in WO 2014/141294. In WO 2014/141294 the step to introduce the bis(2-chloroethyl) group into the molecule comprises conversion of a primary phenyl amine to a tertiary phenyl amine diol, by reaction with ethylene oxide gas. This gives a 52.6% yield. The amine diol is then converted to a bis(2-chloroethyl) phenylamine by reaction with phosphoryl chloride. Using ethylene oxide, or chloroethanol, to convert an aromatic amine to the corresponding bis-(2-hydroxyethyl) amine, followed by chlorination of that intermediate, is a common technique for producing aromatic bis-(2-chloroethyl) amines. It is also known to start from a chloroarene and let it undergo a SNAr-reaction with diethanolamine. The present inventors have applied those methods to produce melflufen (in its salt form), shown in Scheme 2 below.

The inventors have found that using ethylene oxide in THF (route (a) of Scheme 2), no alkylation occurs at 55° C.; increasing the temperature to 60° C. lead to the dialkylated intermediate being formed, but the reaction was very slow. To increase yield and reaction rate the reaction would require high temperatures, but this would cause increased pressure so that the reaction would need be performed in a pressure reactor. Such conditions are likely lead to formation of side products. Similar reaction conditions but using a 50:50 mixture of ethylene oxide and acetic acid (route (b) of Scheme 2) lead to faster reaction times but formation of side products. Using potassium carbonate and chloroethanol (route (c) of Scheme 2) also lead to formation of side product, possibly due to the chloroethanol undergoing partial trans-esterification with the ethyl ester.
The inventors also attempted chlorination of the di-alkylated compound. Chlorination of the bis-(2-hydroxyethyl) compound (4) of Scheme 2 using thionyl chloride in dichloromethane led to significant de-protected side product formation. Chlorination of the bis-(2-hydroxyethyl) compound (4) of Scheme 2 using POCl3 required high temperature and long reaction times. In addition, both thionyl chloride and POCl3 are challenging to handle at large scale due to safety concerns. The inventors also converted the bis-(2-hydroxyethyl) compound (4) of Scheme 2 to the corresponding dimesylate by treatment with methanesulfonyl chloride and triethylamine. The dimesylate was treated then with sodium chloride in DMF at 120° C. However, the crude product of this reaction contained significant side products making this route unsuitable to be used economically at scale.
In summary, none of these routes were found to be suitable for large scale production of high purity melflufen. They do not work well for the synthesis of melflufen, resulting in poor yields and are inefficient. Further, the routes shown in Scheme 2 require multiple steps to form the N, N-bis-chloroethyl amine and use toxic reagents.
The present inventors have discovered an improved process for the production of melflufen (in particular melflufen in the form of its hydrochloride salt), which provides the compound in an excellent yield and with a very high level of purity.